Group A Streptococcus (GAS, S. pyogenes), which is responsible for ~500 million cases of strep throat annually, is also capable of causing severe invasive diseases (10,000 US cases annually) with high mortality rates (>30%), such as streptococcal toxic shock syndrome (STSS). Untreated GAS infection can also lead to serious autoimmune sequelae, including acute rheumatic fever. The M protein, the major surface-associated virulence factor of GAS, has a significant role in each of these disease processes. The M protein impairs phagocytic clearance of GAS by recruiting specific host factors to the GAS surface, the most common being fibrinogen (Fg) and C4b-binding protein (C4BP), that interfere with the deposition of opsonic antibodies and the activation of complement. Furthermore, the M1 type of M protein, after proteolytic release from the GAS surface, forms a proinflammatory complex with fibrinogen that contributes to STSS. Capitalizing on our recent structural discoveries on the M1 protein and its interactions with Fg, the goals of the project are to define the rules governing the interactions of various M protein types with Fg and with C4BP. In vitro and in vivo experiments will be carried out to determine how the M1 protein forms a pathogenic complex with Fg (Aim 1); whether other M types form pathogenic complexes with Fg, and if they do so, how this occurs (Aim 2); and how a remarkably broad variety of M protein types recruit C4BP to the GAS surface for protection through a region that is non-conserved and lacks apparent sequence homology (Aim 3). Once completed, these studies will provide essential knowledge applicable to the design of novel therapeutics or preventive strategies against GAS. This multi-PI proposal draws on the distinct yet highly complementary sorts of expertise in the Ghosh and Nizet laboratories. The Ghosh laboratory has extensive experience with the biochemistry and structural biology of bacterial virulence factors, and the Nizet laboratory has extensive experience in the molecular genetics of group A Streptococcus (GAS) and the study of GAS interactions with host innate immunity in vitro and in vivo. This five-year project will extend a successful approach of joint mentorship and collaboration that has yielded two manuscripts describing the crystal structures of M1 protein and the M1 protein-fibrinogen complex, and another joint manuscript detailing novel mechanisms by which M1 protein promotes GAS resistance to host neutrophil killing. Dr. Ghosh will directly supervise three postdoctoral researchers focusing on biochemical and structural approaches, and Dr. Nizet will directly supervise one postdoctoral researcher focusing on microbiological and immunological approaches.